1. Field of the Invention
The present invention relates to a novel microbial strain capable of degrading organic compounds, and to a process for degrading organic compounds and remedying environment using the same.
The present invention also relates to a process for obtaining a microorganism constitutively expressing oxygenase, and remedying environment using the microorganism obtained thereby.
The present invention also relates to a process for detecting a microorganism constitutively expressing oxygenase.
2. Related Background Art
Recently, a great problem has been an environmental pollution by chlorinated organic compounds which are harmful to organisms and difficult to degrade. Especially the soil in the manufacturing area of paper and pulp industry and semiconductor industry in Japan as well as abroad is considered to be contaminated with chlorinated organic compounds such as tetrachloroethylene (PCE), trichloroethylene (TCE), dichloroethylene (DCE) and the like. Actually there have been many reports on detection of such chlorinated organic compounds through environmental surveys.
It is supposed that chlorinated organic compounds remaining in soil dissolves in groundwater via rainwater etc. thus spread over the area. There is a strong suspicion that these compounds are carcinogens, and further, these are quite stable in the environment; therefore contamination of groundwater, which is used as a source of drinking water, is a serious social problem.
Thus purification of an aqueous medium such as groundwater, soil, and gas phase surrounding them to remove and degrade chlorinated organic compounds, is a very important problem in view of the protection of environment, and techniques needed for the purification have been sought to develop.
Recently, microbial degradation of these compounds has been reported as one of the techniques for purifying the environment contaminated with such chlorinated organic compounds, and various studies have started to put the process to a practical use. The advantages of the biodegradation process utilizing a microorganism includes degradation of chlorinated organic compounds into harmless substances by using an appropriately selected microorganism, no requirement for any special chemicals, and reduction of the labor and costs of maintenance.
The examples of strains capable of degrading TCE are given as follows:
Welchia alkenophila sero 5 (U.S. Pat. No. 4,877,736, ATCC 53570), Welchia alkenophila sero 33 (U.S. Pat. No. 4,877,736, ATCC 53571), Methylocystis sp. strain M (Agric. Biol. Chem., 53, 2903 (1989), Biosci. Biotech. Biochem., 56, 486 (1992), ibid. 56, 736 (1992)), Methylosinus trichosporium OB3b (Am. Chem. Soc. Natl. Meet. Dev. Environ. Microbiol., 29, 365(1989), Appl. Environ. Microbiol., 55, 3155 (1989), Appl. Biochem. Biotechnol., 28, 877 (1991), Japanese Laid-Open Patent Application (JPUPA) 02-92274, JPUPA 03-292970), Methylomonas sp. MM2 (Appl. Environ. Microbiol., 57, 236 (1991)), Alcaligenes denitrificans ssp. xylosoxidans JE75 (Arch. microbiol., 154, 410 (1990)), Alcaligenes eutrophus JMP134 (Appl. Environ. Microbiol., 56, 1179 (1990)), Mycobacterium vaccae JOB5 (J. Gen. Microbiol., 82, 163 (1974), Appl. Environ. Microbiol., 55, 2960 (1989), ATCC 29678), Pseudomonas putida BH (Journal of Japan Sewage Work Assosiation, 24, 27 (1987)), Acinetobactor sp. strain G4 (Appl. Environ. Microbiol., 52, 383 (1986), ibid. 53, 949(1987), ibid. 54, 951 (1989), ibid. 56, 276(1990), ibid. 57, 193 (1991), U.S. Pat. No. 4,925,802, ATCC 53617, this strain was originally classified as Pseudomonas cepacia then classified into Acinetobactor sp.), Pseudomonas mendocina KR-1 (Bio/Technol., 7, 282 (1989)), Pseudomonas putida F1 (Appl. Environ. Microbiol., 54, 1703 (1988), ibid. 54, 2578 (1988)), Pseudomonas fluorescens PFL12 (Appl. Environ. Microbiol., 54, 2578 (1988)), Pseudomonas putida KWI-9 (JPUPA 06-70753), Pseudomonas cepacia KK01 (JPUPA 06-22769), Pseudomonas sp. (JPUPA 02-273599), Nitrosomonas europaea (Appl. Environ. Microbiol., 56, 1169 (1990)), Lactobacillus vaginalis sp. nov (Int. J. Syst. Bacteriol., 39, 368 (1989), ATCC 49540) and so on.
All these strains, however, require a chemical substance such as aromatic compounds and methane as an inducer in order to express their TCE degrading activity.
For example, when the above microorganisms are used for degrading TCE, aromatic compounds such as phenol and toluene are very effective as an inducer. On the other hand, such compounds themselves are contaminants to environment; therefore considering the release into the environment, a complicated operation and monitoring are required. Methane can also serve as an effective inducer; but it is dangerous and difficult to introduce and control it in the environment because it is a flammable gas. Further, the inefficient degradation is another problem because the competitive antagonism occurs between the inducer and the substance subjected to degradation. The same problem occurs when chlorinated aromatic compounds such as PCP and PCB are degraded using a microorganism. The competitive antagonism also occurs between phenol and PCP, and biphenyl and PCB.
To overcome the above problems, Nelson et al. developed a process for degrading chlorinated organic compounds which uses tryptophan, an amino acid, as an inducer (Japanese Patent Application Laid-Open No.4-502277).
Although use of tryptophan allows us to avoid the problem of the toxicity and danger of the inducer itself, tryptophan is very expensive, and the intricacy of introduction and following control of a specific substance in the environment still remains.
Further, the enzyme activity of a TCE degrading enzyme such as oxygenase, expressed with an inducer, is normally maintained for only several hours to one day, and after that, successive addition of the inducer is required, which may cause competitive inhibition in TCE degradation.
Attempts have been made to introduce into a host bacterium a plasmid containing a DNA fragment encoding oxygenase, a TCE degrading enzyme, so that the bacterium will express TCE degrading activity with a harmless inducer or constitutively express it without any inducer. The bacterial strain from which the DNA fragment originates includes Pseudomonas mendocina KR-1 (Japanese Patent Application Laid-Open No.2-503866), Pseudomonas putida KWI-9 (Japanese Patent Application Laid-Open No.6-105691) and Pseudomonas putida BH (Proceedings of the 3rd Meeting of Assembly for study of groundwater/soil contamination and of countermeasures against it, p.213 (1994)).
There are various problems for such a recombinant, for example, requirement for IPTG (isopropyl thiogalactopyranoside), a very expensive substance, as an inducer, or insufficient stability of the plasmid in the host strain. Furthermore, from the viewpoint of public acceptance, release of a recombinant strain into an environment will be inevitably restricted.
Seales et al. made another attempt, that is, Acinetobactor sp. G4 (reclassified from Pseudomonas cepacia G4 to Acinetobactor sp. strain G4 when deposited in ATCC) was mutagenized using a transposon to obtain a strain constitutively expressing oxygenase necessary for TCE degradation (Appl. Environ. Microbiol., 58, p.3977 (1992), WO 92/19738).
This mutant derived from strain G4, however, does not have a sufficient TCE degradation activity, and its stability is questionable because of the transposon. Further, the transposon itself contains resistant markers such as kanamycin resistance, which causes a problem that it may undesirably affect other microorganisms through horizontal transfer when it is released into an environment.
As mentioned above, conventional microorganisms which require no inducer for degrading activity had to be selected by skilled experts using expensive equipments, and they are still not satisfactory in the degradation activity of contaminants.